Large-conductance Ca2+- and voltage-gated K+ (Slo1 BK) channels play numerous physiological and pathophysiological roles and their allosteric gating mechanism is subject to modulation by a variety of cellular signaling pathways. Increasing evidence suggests that certain lipids may serve as signaling molecules. We propose to reveal the biophysical and physicochemical mechanisms of modulation of Slo1 BK channels by two lipid messengers, phosphatidylinositol 4,5-bisphosphate (PIP2) and docosahexaenoic acid (DHA), an omega-3 long-chain polyunsaturated fatty acid enriched in oily fish. The effect of PIP2 on the Slo1 BK channel was reported recently but the mechanism is only poorly known. We will fill this critical knowledge gap by performing thorough mechanistic electrophysiological measurements. Furthermore, we will identify the structural determinants of the auxiliary beta subunit important for the action of PIP2. Our electrophysiological measurements will be complemented with measurements of tryptophan fluorescence of the isolate and purified Slo1 gating ring protein. The biophysical and physicochemical mechanisms of the action of DHA, an emerging lipid messenger, on the Slo1 channel will be also investigated similarly using the electrophysiological and fluorescence methods. The research outcome is expected to provide definitive mechanisms of the PIP and DHA actions on the allosteric gating mechanism of the Slo1 channel and establish the novel paradigm that the Slo1 channel is an omega-3 fatty acid receptor. The modulation of the Slo1 BK channel by DHA may underlie the health-promoting effects of omega-3 long-chain fatty acids. PUBLIC HEALTH RELEVANCE: Nerve/muscle excitability is controlled by a class of proteins called ion channels. Among them, large-conductance calcium- and voltage-gated potassium channels, known as Slo1 BK channels, play particularly important roles by protecting cells from over-excitation. Dysregulation of Slo1 BK channels is implicated in numerous neurological and cardiovascular diseases including epilepsy, hypertension, and delayed cerebral vasospasm, and the channels represent a good therapeutic target. Studies suggest that certain fats, such as phosphatidylinositol 4,5-bisphosphate, have cell signaling roles and others, such as omega-3 long-chain polyunsaturated fatty acids found in oily fish, may exert health-promoting effects. The research program proposed here will attempt to elucidate the mechanisms of the actions of the two lipids on Slo1 BK channels. The anticipated outcome of the research program will advance of our understanding of the health-protective roles of activation of Slo1 BK channels by lipid messengers and may provide a mechanism underlying health-promoting effects of omega-3 fatty acids found in oily fish.